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Forum: Illumina/Solexa 08-12-2019, 12:08 PM
Replies: 3
Views: 251
Posted By Bukowski
Duplex sequencing is obviously better, but...

Duplex sequencing is obviously better, but requires the use of MIDs. Without MIDs there is no way to assign which half of the duplex you're sequencing with a standard library prep and Illumina...
Forum: Illumina/Solexa 08-11-2019, 08:36 AM
Replies: 3
Views: 251
Posted By Bukowski
Q1: Watch this:...

Q1: Watch this: https://www.youtube.com/watch?v=fCd6B5HRaZ8

Q2: There is no requirement for your probes to be dsDNA. Why are you so interested in capturing both strands? They have exactly the same...
Forum: Ion Torrent 08-02-2019, 02:30 PM
Replies: 18
Views: 987
Posted By Bukowski
No one digs through your post history to answer...

No one digs through your post history to answer your questions.

The Ion Torrent workflow is notoriously finicky. Many people submit DNA or pre-made libraries to service providers in other...
Forum: Illumina/Solexa 06-14-2019, 04:08 AM
Replies: 2
Views: 708
Posted By Bukowski
If you look at the handbook you will see that it...

If you look at the handbook you will see that it is for Illumina instruments.
Forum: Ion Torrent 06-14-2019, 04:00 AM
Replies: 2
Views: 806
Posted By Bukowski
I'm not sure the PII chip ever saw the light of...

I'm not sure the PII chip ever saw the light of day. The platform was effectively discontinued with the launch of the S5:
...
Forum: Illumina/Solexa 05-30-2019, 04:20 AM
Replies: 1
Views: 523
Posted By Bukowski
If I did I'd be writing useless 50 page reports...

If I did I'd be writing useless 50 page reports full of terrible graphs and charging $5000 a copy...
Forum: RNA Sequencing 05-05-2019, 06:20 AM
Replies: 2
Views: 684
Posted By Bukowski
How many groups in the experiment? How many...

How many groups in the experiment? How many samples per group? Are they paired samples (i.e. pre and post treatment?) What's the actual experimental setup? What question are you trying to answer?...
Forum: RNA Sequencing 04-17-2019, 03:48 AM
Replies: 1
Views: 760
Posted By Bukowski
Having seen a head to head (some time ago)...

Having seen a head to head (some time ago) between multiple 'stranded' RNA library kits, I can attest that there are degrees of how good they are, running all the way from 'not stranded at all' to...
Forum: Illumina/Solexa 04-01-2019, 03:28 AM
Replies: 5
Views: 769
Posted By Bukowski
People have been doing similar things on the...

People have been doing similar things on the HiSeq too - but the issue here isn't whether it's technically feasible to hack the kits, it's whether your application will be able to cope with the awful...
Forum: RNA Sequencing 03-26-2019, 09:09 AM
Replies: 4
Views: 1,104
Posted By Bukowski
It sounds like your best bet is just doing things...

It sounds like your best bet is just doing things in an embarrassingly parallel manner which is what you're currently doing. I may have misinterpreted your original request, though but the short...
Forum: Pacific Biosciences 03-19-2019, 05:12 AM
Replies: 4
Views: 924
Posted By Bukowski
One of them is an optical platform doing laser...

One of them is an optical platform doing laser based detection of nanometre scale wells, and the other ... isn't. You can't 'skip the details' here, the details are the reason ;)
Forum: RNA Sequencing 03-15-2019, 04:28 AM
Replies: 4
Views: 1,104
Posted By Bukowski
I wouldn't have thought so. You require all the...

I wouldn't have thought so. You require all the reads to assemble the genome, so splitting this across a cluster, without a shared/distributed memory model, doesn't fit the assembly paradigm which is...
Forum: Illumina/Solexa 03-13-2019, 04:19 AM
Replies: 3
Views: 490
Posted By Bukowski
short answer: yes

short answer: yes
Forum: Illumina/Solexa 03-07-2019, 03:26 AM
Replies: 3
Views: 530
Posted By Bukowski
It kind of sounds like your sequencing provider...

It kind of sounds like your sequencing provider hasn't bothered to demux on the dual index.

You might be able to use bbtools Demuxbyname.sh

http://seqanswers.com/forums/showthread.php?t=58221
...
Forum: RNA Sequencing 02-27-2019, 05:33 AM
Replies: 2
Views: 989
Posted By Bukowski
I don't think they say it's only good for...

I don't think they say it's only good for eukaryotes, just that it mimics the nature of eukaryotic transcripts in terms of nucleotide content, size distribution, polyadenylation etc. I think the...
Forum: Illumina/Solexa 02-19-2019, 03:53 AM
Replies: 4
Views: 708
Posted By Bukowski
Not necessarily, the clustering of the flowcells...

Not necessarily, the clustering of the flowcells is completely different on a NovaSeq to a MiSeq - so if they think the pool is balanced on the MiSeq, I'd certainly be looking at the size...
Forum: Illumina/Solexa 02-11-2019, 03:16 AM
Replies: 2
Views: 585
Posted By Bukowski
Maybe. There is not enough data here to answer...

Maybe. There is not enough data here to answer the question. How did you determine the expected genome size? What is the variation in known genome sizes?

Have a look at:
...
Forum: Illumina/Solexa 02-06-2019, 04:18 AM
Replies: 2
Views: 816
Posted By Bukowski
I think you'll find this has been answered...

I think you'll find this has been answered before:

http://seqanswers.com/forums/showthread.php?t=75899
Forum: Bioinformatics 01-30-2019, 06:50 AM
Replies: 1
Views: 460
Posted By Bukowski
People who don't understand the value of a...

People who don't understand the value of a bioinformatician.
Forum: RNA Sequencing 01-21-2019, 01:30 AM
Replies: 8
Views: 1,609
Posted By Bukowski
I just looked at the GenePattern workflow. I can...

I just looked at the GenePattern workflow. I can think of a number of reasons why this might be happening still, but this workflow is EXTREMELY dated. It uses Tophat, which was superseded by Tophat2,...
Forum: Core Facilities 01-19-2019, 07:31 AM
Replies: 3
Views: 1,525
Posted By Bukowski
Of course you can submit cDNA for sequencing, at...

Of course you can submit cDNA for sequencing, at this point it's just 'DNA'. Most cores would probably prefer to take the RNA because then the library preparation is in their hands, and not yours -...
Forum: Pacific Biosciences 01-08-2019, 03:23 AM
Replies: 5
Views: 1,701
Posted By Bukowski
Judging from the total size of the contigs that...

Judging from the total size of the contigs that would be my suspicion, because it looks like you have the whole 5.5Mb there in some form - just not in a single contig (which you can't guarantee, even...
Forum: RNA Sequencing 01-07-2019, 11:35 PM
Replies: 8
Views: 1,609
Posted By Bukowski
IGV would be fine - I'm just interested in...

IGV would be fine - I'm just interested in whether there are any reads mapping to that region
Forum: RNA Sequencing 01-07-2019, 04:29 AM
Replies: 8
Views: 1,609
Posted By Bukowski
So have you visualised the read aligments in the...

So have you visualised the read aligments in the region in the patients?
Forum: RNA Sequencing 01-07-2019, 03:28 AM
Replies: 8
Views: 1,609
Posted By Bukowski
Far too many reasons to possibly speculate on...

Far too many reasons to possibly speculate on from incorrectly running the analysis, to low read coverage in your patient samples to QC differences between the samples to differences is sample...
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